Characterization of the role of the Burkholderia pseudomallei type 3 secretion system using in vivo imaging.

Burkholderia pseudomallei;melioidosis;T3SS;IMIT

Maria Gabriela Gutierrez

University:University of Louisville

Department:Microbiology and Immunology

关键词: Burkholderia pseudomallei;    melioidosis;    T3SS;    IMIT;   

Others  :  https://ir.library.louisville.edu/cgi/viewcontent.cgi?article=3385&context=etd

美国|英语

来源: The Universite of Louisville's Institutional Repository

【 摘 要 】

Melioidosis is a fatal infectious disease caused by the Tier 1 Select Agent Burkholderia pseudomallei. Hallmarks of melioidosis include pneumonic disease and prominent septicaemic spread. Both forms of disease are contingent upon the bacterium’s intracellular life cycle and particularly on its ability to escape from host cell phagosomes. Upon encountering a host cell, B. pseudomallei is internalized into membrane-bound vacuoles from which the bacterium must rapidly escape to the cytoplasm in order to replicate and promote its survival. In the host cytoplasm, B. pseudomallei is capable of polymerizing actin for intracellular and intercellular motility and spread, lysing the host cell and perpetuating the cycle of infection. Commonly used intranasal and aerosol models to study respiratory melioidosis result in significant upper respiratory tract colonization, dramatically altering disease progression. Accordingly, we developed an improved lung-specific instillation approach to deliver bacteria directly into mice lungs, coupled with in vivo optical imaging and observed the development of disease that closely resembles human melioidosis in mice. We found that in the absence of upper respiratory tract infection, a capsular polysaccharide (CPS) mutant is only 6.8-fold attenuated. This mutant is unable to spread to secondary sites of infection, consistent with the role of capsule in protecting the bacterium from host antimicrobial activity. Similarly, a type 3 secretion system cluster 3 (T3SS3) structural mutant is spread deficient, yet this mutant is attenuated 290-fold, strongly suggesting that T3SS3 is critical for respiratory melioidosis. Having a strong platform for studying the pathogenesis of B. pseudomallei in a mouse model of lung-specific melioidosis, we used transposon mutagenesis to comprehensively identify virulence factors required for B. pseudomallei lung colonization and spread to the liver and spleen. Notably, T3SS3, capsular polysaccharide and type 6 secretion system cluster 5 (T6SS5) were the major genetic loci required for respiratory melioidosis. A T6SS5 mutant is not attenuated by LD50 estimations using our lung-specific melioidosis mouse model. Yet by competition analysis T6SS5, T3SS3 and CPS mutants were attenuated, substantiating the requirement of these factors for B. pseudomallei infection as previously reported. These results highlight the importance of competition analysis for studying the fitness of distinct virulence determinants. Importantly, T3SS3 was the only virulence determinant attenuated by both LD50 analysis and competition studies, corroborating the critical requirement of this virulence system for respiratory melioidosis. B. pseudomallei is a facultative intracellular cytosolic bacterium and its ability to survive intracellularly is fundamental to mammalian host infection. Upon B. pseudomallei